We shall continue to isolate, map and characterize mutants which have altered virulence properties. These mutants will be generated either by inserting transposition elements, such as Tn5 or Tn3, through the techniques of site specific mutagenesis and marker exchange, or through the use of mutants which have suffered deletions either in vitro or in vivo. From such an analysis we will be able to map mutation genes on the tumor inducing plasmid which give an altered tumor phenotype. Sine we are able to introduce Ti-plasmid DNA into plant protoplasts which can be regenerated into intact plants, it should be possible to determine whether mutations which map outside the region transferred into the plant are virulent in the protoplast system. Further, we will determine if various regions of the cloned Ti-plasmid are able to transform protoplasts. Another major effort will be directed at understanding the regulation of gene expression of the T-region in Agrobacterium and in the tumor. Thus, we will determine whether the same transcripts are present, whether there is any evidence for processing of the transcripts and specifically why octopine synthesis is not expressed in Agrobacterium but is expressed in the tumor. All of these studies will contribute to our understanding of the mechanism by which procaryotic DNA transforms eucaryotic cells.